Paclitaxel has shown remarkable efficacy in the treatment of cancer, but the emergence of drug resistant tumor cells limits its ability to cure disease. The long term goal of the studies described here is to define the molecular mechanisms of drug resistance. Destabilization of microtubule assembly by mutations in tubulin represents a major mechanism of resistance to paclitaxel. Preliminary studies indicate that these mutations alter 3 closely spaced leucine residues in beta-tubulin. To understand this mechanism at a detailed molecular level, the following questions will be addressed: 1) Do all paclitaxel resistance mutations map to the same area of beta-tubulin and do mutations in alpha-tubulin also cluster in one area of the gene? Tubulin genes from existing paclitaxel resistant CHO cells will be sequenced, and additional mutants will be created by transfecting wild-type cells with randomly mutagenized alpha- and beta-tubulin cDNAs. 2) Why are only a limited set of amino acid residues involved in conferring resistance to paclitaxel? Site-directed mutagenesis, transfection, and assays for tubulin assembly and paclitaxel resistance will be used to test the tolerance for small vs. large, hydrophobic vs. hydrophilic, and charged vs. uncharged substitutions at the 3 altered leucine and at other adjacent amino acid residues. 3) What is the topological and functional significance of this region? The 3 altered leucines primarily affect a loop connecting helices 6 and 7 of beta-tubulin. Second-site suppressor analysis and chemical crosslinking will be used to determine what interacts with this loop. Antibodies, fluorescent reporters, and inhibitory peptides will be used to determine whether the loop becomes buried when microtubules assemble and whether site-specific agents can inhibit assembly in vitro. 4) Can principles derived from studying one cell type be extrapolated to other cell types? The role of beta-tubulin isotopes in drug resistance will be studied using site-directed mutagenesis and transfection of different isotypes. Also, the frequencies of different resistance mechanisms will be tested in human cell lines surviving paclitaxel treatment. The studies in this proposal will elucidate the mechanism of action of paclitaxel and define a major mechanism by which cells can escape the cytotoxicity of the drug. This knowledge should lead to better paclitaxel analogs, to improved strategies for circumventing paclitaxel resistance, and to the development of diagnostic tests for the detection of paclitaxel resistant tumor cells.